| Acknowledgements | 第1-9页 |
| Abstract | 第9-17页 |
| 中文摘要 | 第17-20页 |
| Table of Contents | 第20-26页 |
| General Introduction | 第26-33页 |
| Chapter 1 CLONING, SEQUENCING^ EXPRESSION AND PURIFICATION OFLeptospira interrogans rColA PROTEIN | 第33-56页 |
| ·Materials and Methods | 第33-43页 |
| ·Bacterial strains and plasmids | 第33页 |
| ·Reagents | 第33-34页 |
| ·PCR Primers | 第34页 |
| ·Preparation of the L.interrogabs genomic DNA for PCR | 第34-38页 |
| ·Amplification of colA gene and identification of PCR products | 第38页 |
| ·Cloning of the colA gene | 第38-39页 |
| ·Sequencing and comparison of clones | 第39页 |
| ·Construction of recombinant bacterial | 第39-40页 |
| ·Induction of recombinant bacterial | 第40页 |
| ·Procaryitic expression and purification of ColA protein | 第40-42页 |
| ·Molecular mass determination and preparation of antiresum against rColA | 第42-43页 |
| ·Results | 第43-55页 |
| ·Amplification of colA gene and restriction endonucleases digest of recombinant T-vector | 第43页 |
| ·Sequencing of the colA gene clones | 第43页 |
| ·Identification of recombinant plasmids expressing rColA | 第43-44页 |
| ·Expression, identification and molecular mass of the purified ColA protein | 第44-55页 |
| ·Discussion | 第55-56页 |
| Chapter 2 ENZYMATIC ESSAY OF COLLAGENASE ENCODED BY colAGENE FROM Leptospira interrogans AND IT'S FEATURES | 第56-71页 |
| ·Materials and Methods | 第56-60页 |
| ·Substrates and Reagents | 第56-57页 |
| ·Collagenase enzymatic assay | 第57-60页 |
| ·Quantitative enzymatic essay | 第57页 |
| ·Quanlitative enzymatic essay | 第57-60页 |
| ·Results | 第60-68页 |
| ·Quantitative enzymatic essay:leptopspiral collagenase digested collagen | 第60-61页 |
| ·Substrate specificity for collagenase activity | 第61页 |
| ·Specificity of different pH,ions and buffer systems for Collagenas eactivity | 第61-62页 |
| ·Kinetic:apparent Michaelis-Menten constant(Km)and substrate turnover number(Vmax) | 第62-68页 |
| ·Discussion | 第68-71页 |
| Chapter 3 MEASUREMENT OF mRNA LEVEL OF colA GENE OF L.interrogans AND ColA PROTEIN EXPRESSION AND SECRETIONDURING INFECTION WITH HUVEC OR HEK 293 CELLS | 第71-83页 |
| ·Materials and Methods | 第71-78页 |
| ·Baterial strains and culture conditions | 第71页 |
| ·Reagents | 第71-72页 |
| ·Primers for Real Time PCR | 第72页 |
| ·Bacterial and Cells culture | 第72-73页 |
| ·Infection of Leptospires with HUVEC or HEK 293 cells | 第73页 |
| ·Extraction of the mRNA and their conversion to cDNA | 第73-75页 |
| ·Real Time PCR | 第75-76页 |
| ·Extraction of the total proteins from the infected leptospires and detection of the expressed Col A protein after infection by Western-Blot | 第76-77页 |
| ·Extraction of the proteins from the supernatant and detection of the secreted ColA protein after infection by Western-Blot | 第77-78页 |
| ·Results | 第78-81页 |
| ·Transient transcription and expression of the leptospiral collagenase during infection with HUVEC or HEK 293 cells | 第78-79页 |
| ·Secretion of the leptospiral collagenase during infection with HUVEC or HEK 293 cells | 第79-81页 |
| ·Discussion | 第81-83页 |
| Chapter 4 THE PIVOTAL VIRULENCE ROLE IN PATHOGENESIS OFLeptospira COLLAGENASE ENCODED BY colAGENE IN HAMSTERS | 第83-116页 |
| ·Generation of col A gene knock-out mutant(colA mutant) | 第83-95页 |
| ·Baterial strains and plasmids | 第83-84页 |
| ·Reagents | 第84页 |
| ·Primers for PCR | 第84-85页 |
| ·Preparation of L. interrogans Genomic DNA for PCR | 第85-88页 |
| ·Amplification of colA gene and identification of PCR products | 第88-89页 |
| ·Cloning of the colA gene | 第89页 |
| ·Sequencing and comparison | 第89-90页 |
| ·Construction of the recombinant suicide plasmid pUC 19~(kan-colA—) | 第90-93页 |
| ·Electro-transformation of recombinant suicide plasmid in L. interrogans strain Lai, its culture and identification | 第93-94页 |
| ·Sequencing of the recombinant bacterial | 第94-95页 |
| ·Animal tests:Infection of hamsters with colA~- mutant or wild-type leptospires | 第95-96页 |
| ·Results | 第96-113页 |
| ·Sequencing results of colA gene,kan cassette and suicide recombinant plasmid pUC19~(kan-colA~-) | 第96页 |
| ·Characterization of the colA~- mutant | 第96-97页 |
| ·Attenuated virulence of the colA~- mutant in hamsters | 第97-113页 |
| ·Discussion | 第113-116页 |
| Chapter 5 | 第116-121页 |
| ·General Conclusion | 第116-119页 |
| ·Potential for future development of the study | 第119-121页 |
| Chapiter 6 Brief Summary of the geographic distribution of Leptospirosis inthe world and in P.R.China | 第121-128页 |
| ·Introduction | 第121-122页 |
| ·Leptospirosis and geographic distribution | 第122-128页 |
| ·Leptospirosis:Geographic distribution in the wold | 第122-125页 |
| ·Leptospirosis in People's Republic of China | 第125-128页 |
| REFERENCES | 第128-136页 |
| Scientiifc research achievements of the thesis writer | 第136页 |
